INSTITUTE OF BIOMEDICAL ENGINEERING & TECHNOLOGY

LIAQUAT UNIVERSITY OF MEDICAL AND HEALTH SCIENCES, JAMSHORO

Subject: Biomedical Instrumentation II Batch: 21 BME Year: 3rd Term: 6th
Open-ended Lab # 02

Name: ______________________________________________Roll No: ____________________

Signature of Tutor: ______________________________ Date:___________________________

Objective: To get the understanding of The polymerase chain reaction (PCR)

Introduction: The polymerase chain reaction (PCR) is a very important tool in molecular biology
because it enables a sequence of DNA to be replicated across several orders of magnitude,
generating millions to billions of copies by cycling the reagents (DNA, Primers, dNTPs, enzymes,
etc.) through the prescribed temperature regimes. The idea is very simple: DNA, which is double
stranded helix of nucleotides (DNA building blocks), is heated to ~ 95°C where it unwinds into
two single DNA strands. The temperature is then lowered to ~ 50 – 60 °C, where target specific
DNA primers anneal themselves onto the two single stranded template DNA molecules. Finally the
temperature is again raised to ~72 °C where DNA polymerase enzyme assembles a new double
stranded DNA from nucleotides, by using the single stranded DNA as a template. After one cycle
(see fig a. below), two DNA molecules are produced and in subsequent cycles the number of DNA
molecules doubles in a geometric progression (2N after N cycles), exponentially increasing the
concentration of target DNA.

Apparatus:
Conventional Thermo-Cycler
Convective flow Thermo-Cycler

The process of heating and

cooling the PCR solution is
known as thermal cycling.
Conventionally thermal
cycling is performed by programmed heating
and cooling of heavy metallic blocks containing
the PCR reaction mixture in thermo-cyclers.
These are not very efficient as they consume
large reaction time, sample volume and energy.
 This lab has developed a novel technique to perform PCR in microfluidic convective cells. It is a
simple design where the PCR mixture is confined in a cylindrical enclosure whose bottom
temperature is maintained at a higher temperature than the top. This causes the fluid to circulate
through the cell; shuffling the PCR reagents through the optimum temperature regimes (see fig b).

Procedure
1 a. Conventional thermo-cycler
1. After the reagents have been thawed from their frozen state, pipette
appropriate amounts of each reagent in that sequence into a micro-tube.
Add the polymerase enzyme at the very end.
2. Place the micro tubes with the reagent mixture in the thermo-cycler wells
and follow the following thermo-cycling.
3. After the thermo-cycling, remove the micro-tubes from the thermo-cycler and
pipette the products out for analysis using gel electrophoresis.
1b. Convective flow thermo-cycler
1. Before loading the reagent mixture, seal the bottom of the convective cells with
thin aluminum sheets. Rinse the cell walls with a 10 mg/ml aqueous solution
of bovine serum albumin followed by Rain-X Anti-Fog. This is done minimize
adhesion of reagents on the cell walls.
2. After the reagents have been thawed from their frozen state, pipette appropriate
amounts of each reagent in that sequence into a micro-tube. Add the polymerase
enzyme at the very end.
3. Transfer the reagents with the help of a micro pipette into the convective cells.
Seal the top of the cell with aluminum sheet and ensure that no air bubbles show
up.
4. Clamp the convective cells between the top and the bottom plates of the convective device tightly and run
the lynntech software through the computer interface to maintain the temperature of the top and bottom
plate as 55 o c and 95 o c respectively. Refer to section lc. below for software operation.
5. After running the reaction for 15 minutes, remove the top aluminum sheet and pipette the products out of
the convective cells into another micro tube
for analysis using gel electrophoresis.

lc. Operating the COM0190 software

in manual mode
 1. Graphical representation of top heater temperature (0C).
2. User input box for setting top heater temperature (0C). Actual temperature (0C) of top heater as
measured by the system.
3. Temperature value (0C) for top heater that the controller board has been programmed to achieve.
4. Test unit numbur with corresponding controller board ID number selected for data display and
control.
5. by the software application.
6. Controller board ID number feedback from the controller board.
7. Operation Mode Selector.
8. Operation controls for running unit in Auto mode.
9. Operation controls for running unit in Manual mode.
10. Controls for logging temperature data.
11. Temperature value (0C) for bottom heater that the controller board has been programmed to
achieve.
12. User input box for setting bottom heater temperature (0C).
13. Actual temperature (0C) of bottom heater as measured by the system.
14. Graphical representation of bottom heater temperature (0C).
15. Displays time of current process the instrument is performing.
16. Displays total time experiment has been running in seconds.

a) Set Operation Mode Selector to "Man" (7). In manual mode the user can set each heater temperature
individually.
b) Input temperature (0C) for single or both heaters in the heater input boxes (2, 12). Press 'Board" (9) to
update temperature for selected test unit. Heaters will heat to the input values and remain at that
temperature until values are changed.
c) Logging temperature data can be performed by the user pressing "Log Data", the box will turn red, and
temperature data will now be logged. To stop the data logging press "Log Data" again.
d) The temperature variation of the two heaters can be viewed under the "Plots" tab.
e) When the operation is completed, set the
Operation Mode Selector to "Off". This will
disable the heaters.

1. Test unit ID and board number ID for plot

data.
2. Channel display selection.
3. Legend display.
4. Graph area.
5. Expand plot button.
6. Tracking enable
Gel electrophoresis analysis
1. Prepare a 2 wt % agarose gel by heating 10 g of agarose with 500 ml of Ix buffer on a
stirring hot plate until the solution becomes clear.
2. Load the agarose gel into the casting tray and insert the comb and let the gel set for 30
minutes.

3. Remove the comb and add Ix TAE buffer until the gel is submerged.
4. Prepare fluorescently stained DNA samples by mixing 2 gl- 100x SYBR Green I solution,
2 gl- PCR product from convective cells/thermo-cycler, 2 ul- 6x orange loading dye and 4
PIL TAE buffer.
5. Add DNA samples into the wells and run the separation at 60 V for 1 h with a 100 bp DNA
ladder sizing marker.
6. Remove the gel and photograph it under UV light to view results.